The present invention relates to an economically beneficial, hence, inexpensive, method which is simple avoiding labor intensive techniques, such as HPLC or column chromatography, for large-scale isolation of Nimbin. Specifically, the invention relates to a method of using a combination of alcohol solubilization, organic partitioning and crystallization to obtain highly purified Nimbin from Neem kernels.
Nim, Neem or Nimba (Melia azadiracta, Melia indica) is native to India and is cultivated to a large extent because of its medicinal properties. These therapeutic properties are so highly valued to the indigenous population that the nim tree plays the role of xe2x80x9cvillage dispensaryxe2x80x9d in many rural areas (Siddiqui and Mitra, J Sci Indus Res (India) 4:5 (1945)). Its twigs are used as a toothbrush for general oral hygiene and the leaves and bark are employed for a variety of uses including wound healing and treatment of various skin ailments (Siddiqui and Mitra, (1945)).
The first report of the pesticidal properties of Neem appeared around 1927 when Mann and Burns (Agr J India (Calcutta) 22:325 (1927)) observed during the locust cycle of 1926-27 that adult locusts did not feed on Neem leaves. This was followed by Chopra (Rept of Dept of Agr (Punjab) Pt. 2, 1:67 (1928)) who treated the extact of Neem leaves as contact poison on grub weevils.
Since then, a number of publications describing the various activities from different parts of the Neem tree have appeared. For example, Neem oil has shown antifeedant activity against Nephotettix virescens (leafhopper of rice). (Neem Newsletter 1:28 (1984)).
Azadirachta Idica (Neem) seed kernels contain the active constituents which have interested a number of workers since Chaterjee and Sen (Ind J Med Res (1920) 8:356) reported the isolation of margosic acid in 1919. The investigation of the bitter principles continued often with conflicting results. (See, e.g., Siddiqui, Curr Sci (India) 11:278 (1942)). In 1923, Wetson et al. (J Soc Chem Ind (1923) 42:387) isolated from the soap lye of the oil, a sulfur-free crystalline acid margoso-picrin (yield 0.12 to 0.017% oil and 0.15 to 0.24% amorphous bitter acids). Sen and Bennerjee (J Ind Chem SOC (1931) 8:773) noted the isolation of a sulfur-containing acidic bitter principle from aqueous extracts of the oil. Quadrat-I-Khuda et al. (J Ind Chem SOC (1940) 17:189) reported the isolation of a sulfur-containing essential oil and an amorphous, water-soluble bitter principle from an aqueous extract of the oil previously distilled to remove steam-volatile products. Industrial methods for isolation of such bitter principles have been reported to give yields of approximately 1.2%.
Neem seeds contain close to 100 molecules of which 84 have been identified. Nimbin was first isolated by Siddiqui in 1942 but its structural elucidation was accomplished by Narayanan et al. (Chem and Ind (1962) 1238; Chem and Ind (1964) 322; Chem and Ind (1964) 324; J Chem (1964) 2:108) and Harris et al. (Tetrahedron (1968) 24:1517) in the sixties. Merck Index (12th ed.) (1996) describes Nimbin as the first bitter principle from various parts of nim tree, Azadirachta indica, Juss., having a melting point of 205xc2x0 C.
On account of the medicinal and antifeedant uses of nim, increased efforts have been made by different investigators to isolate the active ingredients which account for the above properties. In view of the growing importance of Neem kernel and its constituents as commercial products, the present inventors teach a method of isolating one of the active principles in a more efficient and practical manner which comports with industrial production.
The object of the present invention is to provide a simplified method for large-scale extraction of pure Nimbin from Neem kernel powder comprising extracting Neem kernel powder with alcoholic solvents. In a related aspect, the solvents can include methanol, ethanol and isopropanol.
The invention also envisages concentrating the alcoholic extract under vacuum, to include separating sugars and starches by partitioning between water and chlorinated hydrocarbon layers. Such chlorinated hydrocarbons include but are not limited to dichioromethane, dichioroethane and trichloroethylene. The method also comprises filtering the chlorinated hydrocarbon extract, stripping and precipitating the extract with a hydrocarbon solvent (e.g., hexane, heptane, octane). Moreover, the hydrocarbon extract is stripped, suspended and stirred in twice the volume of hydrocarbon. The mixture then can be cleared (e.g., by centrifugation), and the resulting solids suspended in 3 times the volume of alcohol where the material is stirred, centrifuged, dried and re-crystallized twice with alcohol and activated carbon.
Another object of the present invention is to provide a process for the preparation of essentially 98.50% pure Nimbin from pulverized Neem kernels. In a related aspect, the Nimbin obtained by the present method has a melting point of 210-212xc2x0 C. and is tasteless.
The resulting compound may be formulated into many different compositions, such as lotions, sprays, cremes and the like for use on humans, animals and vegetation.
Other features and objects of the present invention will become apparent from the following description.
The method employed for the isolation of the bitter products does not involve the initial saponification of the oil and is based on the difference in the solubilities of the various constituents in diluted alcohol and other non-miscible organic solvents such as ether, petroleum ether and ethyl acetate. Apart from the fact that such a method ensures the isolation of the constituents in a native form, the method has the advantage of yielding a comparatively purified oil which can be used for industrial purposes (e.g., photo-degradation products of Nimbin have insecticidal activity).
According to the invention, Neem kernels are pulverized and extracted with alcoholic solvents including, but not limited to, methanol, ethanol or isopropanol. Methanol is a preferred alcohol. Alcohol is added at approximately 1-5 times the volume by weight of kernel powder at about 25-50xc2x0 C. for between about 2 hours to about 24 hours under agitation. In a preferred embodiment, the ratio of alcohol to kernel powder is 2, 3 or 4, preferably 2.5. In a related aspect, the incubation temperature is about 30xc2x0 C., 40xc2x0 C., 45xc2x0 C. or 47xc2x0 C. In a preferred embodiment, the temperature is 40xc2x0 C. The incubation can occur for 3, 5, 7, 9, 11, 13, 15 or more hours. In a related aspect, longer incubation times are not necessary and 2, 4, 6 or 8 hours of incubation can be sufficient. A preferred incubation range is 4 to 6 hours. In one embodiment, when the incubation temperature is 40xc2x0 C., the temperature is reduced to 30xc2x0 C. before separation of the phases. In a related aspect, the temperature is brought down to about 25xc2x0 C. and treated to separate the liquid and solid phases.
The solid phase can be re-extracted with an excess of about 1.5-4 times the volume of alcohol by original weight of kernel powder under the similar conditions. A preferred alcohol is methanol. It is preferred that the same alcohol is used for both extractions. A preferred ratio of alcohol to kernel powder is 1.5. The solid phase is stripped of solvent and stored.
Both liquid phases are pooled and solvent concentrated to approximately 5% of the original volume. A suitable means of concentration is evaporation, the liquid being passed successively through falling film and wiped film evaporators under reduced pressure (600 mm Hg). The resultant gummy mass containing Neem limonoids in addition to sugars, starches and lipids is partitioned between water and a chlorinated hydrocarbon solvent. Suitable chlorinated hydrocarbon solvents include but are not limited to, methylene dichloride, ethylene dichloride and trichloroethylene. In a preferred embodiment, ethylene dichloride and water are used to extract active principles from Neem kernels. In a related embodiment, aqueous and organic phases are separated, the organic phase is filtered and stripped of solvent under reduced pressure (about 600 mm Hg).
While the thick organic mass is being stirred, 2-10, preferably 4-10 and more preferably, 6-8 times the volume of hydrocarbon solvent (to include but not limited to aliphatic solvents such as hexane, heptane and octane) are added. In a preferred embodiment, hexane is added and stirred. The mixture is transferred to remove the hydrocarbon solvent, for example, under vacuum to a distillation still. The residue, a syrupy mass, contains a mixture of Neem limonoids and lipids from which Nimbin is isolated
The syrupy mass is added to a hydrocarbon solvent (e.g., including, but not limited to, hexane, heptane and octane). In a preferred embodiment, heptane is the solvent. In a related aspect, the hydrocarbon is added at 2-5 times the volume, preferably 2.5 times the volume. A thick mass separates out which is collected by centrifugation. The solid mass then is cleared (e.g., spun) for 15 minutes in the centrifuge and washed with the hydrocarbon solvent. Spinning is continued and the product is unloaded from the centrifuge. In a preferred embodiment, the spring is carried out by centrifugation in an appropriate centrifugation device designed for industrial use/capacity. In a related embodiment, centrifugation speed is between about 1000 and 1600 rpm. The solid phase then is suspended in 3 times w/v of an alcoholic solvent, including, but not limited to, methanol, ethanol and isopropanol. In a preferred embodiment methanol is added, the mixture is stirred for 30 minutes and centrifuged. The product is washed in the centrifuge with the same solvent and dried under vacuum.
The result is technical grade Nimbin of 90%+purity with a melting point of 185-190xc2x0 C. The product is crystallized twice with methanol 1:20 w/v. After the second re-crystallization, Nimbin of essentially 98.5% purity is obtained, which is white, crystalline, tasteless and melts between about 210xc2x0 and about 212xc2x0 C.
The Nimbin of the instant invention has broad spectrum insecticidal action against several classes of insects including but not limited to Lepidoptera such as diamondback moth, imported cabbage worm, cabbage looper, tobacco budworm; Coleoptera such as Colorado potato beetle; Homoplera such as green apple aphid, green peach aphid; and Homoptera such as potato leafhopper.
For the control of insects in agriculture and horticulture the dosage of the composition is calculated based on the concentration of Nimbin in the extract In general, a dosage corresponding to from about 0.1 gram to about 10 kilograms of Nimbin per hectare may be used and from about 5 grams to about 200 grams per hectare of Nimbin is preferred. The exact amount of dosage for a given situation can be routinely determined and depends on a variety of factors, for example, the substance used, the kind of pest, the formulation used, the state of the crop infested with the pest and the prevailing weather conditions. The term xe2x80x9cinsecticidalxe2x80x9d as employed in the specification and claims of the application is to be construed as any means which adversely affects the existence or growth of the target insects. Such means can comprise a complete killing action, eradication, arresting in growth, inhibition, reducing in number or any combination thereof. The term xe2x80x9ccontrolxe2x80x9d as employed in the specification and claims of this application is to be construed as meaning xe2x80x9cinsecticidalxe2x80x9d or protecting plants from insect damage. By xe2x80x9cinsecticidally effective amountxe2x80x9d is meant that dosage of active substance sufficient to exert insect xe2x80x9ccontrol.xe2x80x9d
The Nimbin of the present invention, for practical applications, can be utilized in the form of compositions or formulations. Examples of the preparation of compositions and formulations can be found in the American Chemical Society publication xe2x80x9cPesticidal Formulation Research,xe2x80x9d (1969), Advances in Chemistry Series No. 86, written by Wade Van Valkenburg. In these compositions and formulations, the active substance is mixed with conventional inert agronomically acceptable (i.e., plant compatible and/or pesticidally inert) diluents or extenders such as solid type usable in conventional compositions or formulations as is well known in the art. If desired, adjuvants such as surfactants, stabilizers, antifoam agents and antidrift agents may also be added.
Examples of compositions and formulations according to the invention are those known to one skilled in the art and include aqueous solutions and dispersions, oily solutions and oil dispersions, pastes, dusting powders, wettable powders, emulsifiable concentrates, flowables, granules, baits, invert emulsions, aerosol compositions and fumigating candles.
The compositions and formulations are prepared in a known manner to one skilled in the art, for example by extending the active compounds with conventional dispersible liquid diluent carriers and/or dispersible solid carriers optionally with the use of carrier vehicle assistants, e.g., conventional surface-active agents, including emulsifying agents and/or dispersing agents, whereby, for example, in the case where water is used as diluent, organic solvents may be added as auxiliary solvents.
Adhesives such as caxboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or lattices, such as gum arabic, polyvinyl alcohol, polyvinyl cellulose, and polyvinyl acetate, can be used in the formulations to improve the adherence of this pesticide. Furthermore, a lubricant such as calcium stearate or magnesium stearate may be added to a wettable powder or to a mixture to be granulated.
The Nimbin of the present invention may be employed alone and/or with such solid and/or liquid dispersible carrier vehicles and/or with other known compatible active agents, especially plant protection agents, such as other insecticides, arthropodicides, nematicides, fungicides, bactericides, rodenticides, herbicides, fertilizers, growth-regulating agents, synergists, etc., if desired, or in the form of particular dosage preparations for specific applications made therefrom, such as solutions, emulsions, suspensions, powders, pastes, and granules which are thus ready for use.
As concerns commercially marketed preparations, these generally contemplate carrier composition mixtures in which the Nimbin is present in an amount substantially between about 0.1% and 99% by weight, and preferably between about 1% and 75% by weight, of the mixture. Carrier composition mixtures suitable for direct application in the field generally contemplate those in which the Nimbin present in the extract is used in an amount substantially between about 0.0001% and 5%, preferably between about 0.001% and 3%, by weight of the mixture. Thus the instant invention contemplates overall formulations and compositions which comprise mixtures of a conventional dispersible carrier such as (1) a dispersible inert finely divided carrier solid, and/or (2) a dispersible carrier liquid such as an inert organic solvent and/or water, with or without a surface-active effective amount of a carrier vehicle assistant (e.g., a surface-active agent, such as an emulsifying agent and/or a dispersing agent), and an amount of the Nimbin generally between about 0.0001% and about 99% by weight of the composition, preferably between about 0.001% and about 90% by weight of the composition, and more preferably between about 0.01% and about 75% by weight of the composition which is effective to control insects.
The hydrogenated extracts can be applied as sprays by methods commonly employed, such as conventional high-gallonage hydraulic sprays, low gallonage sprays, ultra low volume sprays, airblast spray, aerial sprays, and dusts.
Furthermore, the present invention contemplates methods of selectively killing, combating or controlling pests, which comprises contacting insects with a correspondingly combative or toxic amount (i.e., an insecticidally effective amount) of the Nimbin of the invention alone or together with a carrier vehicle (composition or formulation) as noted above.
The term xe2x80x9ccontactingxe2x80x9d as employed in the specification and claims of this application is to be construed as applying to at least one of (a) such insects and (b) the corresponding habitat thereof (i.e., the locus to be protected, for example, to a growing crop or an area where a crop is to be grown) the active compound of this invention alone or as a constituent of a composition or formulation.
It will be realized, of course, that the concentration of the particular Nimbin utilized in admixture with the carrier vehicle will depend upon such factors as the type of equipment employed, method of application, area to be treated, types of pests to be controlled and degree of infestation. In addition to the aforementioned ingredients, the preparations according to the invention may also contain other substances commonly used in preparations of this kind.
The compounds of this invention can be administered to hosts for therapeutic applications by oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), nasal, vaginal, rectal, or sublingual routes of administration, and can be formulated in dosage forms appropriate for each route of administration. A preferred route is topical.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. These liquid dosage forms may contain inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.
Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax. Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
Topical preparations can take the form of ointments, salves and creams, for example. Known diluents, suspending agents, amphipathic agents and so on, are known in the art.
The dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient shall be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. Generally dosage levels of between 0.001 to 10 mg/kg of body weight daily are administered to mammals.
For example, pure Nimbin has been consumed orally (10 mg/day) in a test study to observe therapeutic/pharmacological effects of the compound various systems/maladies in humans. In a related aspect, Nimbin has been found to have analgesic effects and topical administration modulates the tone of the skin. Further, systemic administration seems to have regressive actions on benign tumors.
The following illustrates the efficacy and advantages associated with the instant manufacturing process in accordance with the present invention. It is understood that such is for illustration purposes only and that alternative embodiments are contemplated as within the scope of the present invention.